| Summary: | With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC<sub>50</sub> values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD<sub>600</sub> measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against <i>Staphylococcus aureus</i>, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution <i>S. aureus</i> growth inhibition profiling was evaluated on a crude extract of an inactive <i>Alternaria</i> sp., spiked with ciprofloxacin.
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